Activation of mu (MOR) and delta opioid receptors (DOR) produces the motivational behaviors that are concomitant with opiate addiction. The complex neuronal adaptations associated with these behaviors are thought to be mediated through opiate targeting of GABAergic and cholinergic neurons that are either located within the medial prefrontal cortex (mPFC), or in other brain regions that provide major cortical innervation. To determine the cellular sites for opiate modulation of mPFC transmission through activation of MOR and DOR, we will combine dual-labeling electron microscopic immunocytochemistry, retrograde tract-tracing and in vivo intracellular and extracellular single-unit recording in rat brain. Study 1 will test whether MOR and DOR are localized to 1) specific neuronal compartments within GABAergic cells of the mPFC, 2) cell bodies that project directly from the VTA to the mPFC that are known to contain dopamine and possibly GABA, and 3) axon terminals in the VTA that innervate projection neurons to the mPFC. Results from this study will establish potential functional sites for opiate modulation of MPFC transmission through activation of MOR and DOR located on selective sites of local GABAergic neurons or projection cells from the VTA. Study 2 will test the hypotheses that MOR and DOR are located on either 1) cholinergic terminals or their targets within MPFC, or 2) basal forebrain cell bodies that are known to contain acetylcholine, and also project to the MPFC. Results from this study will establish whether opiate modulation of cortical cholinergic transmission is likely to occur through changes in local presynaptic release of acetylcholine, the receptivity of cholinergic targets, or activation of extrinsic projection neurons. Study 3 will test the hypothesis that neurons in the VTA that are physiologically characterized as non-dopaminergic, contain GABA, MOR and/or DOR. This study will establish the neurotransmitter in physiologically identified VTA neurons, and also will determine the functional sites for opiates within putative GABAergic neurons in the VTA. These integrative studies will determine the cellular sites for opiate targeting of MOR and DOR within anatomically and physiologically identified neurons involved in modulation of the MPFC. Results from these studies are important for establishing the disinhibitory circuits involved in the motivational aspects of opiate use. Elucidating the chemical identity of cortical and midbrain targets of opiates that are active at MOR and DOR are crucial for clinical intervention during opiate craving and withdrawal.